Oleophilic biodegrading additive and method of treating hybrocarbon polluted medium

ABSTRACT

Biodegradation additive characterized in that it consists of a mixture of (i) at least one assimilable nitrogen source composed of at least one unsubstituted or substituted amino acid; (ii) at least one phosphorus source, the ratio of nitrogen to phosphorus ranging from 2 to 100, said additive being subjected to a treatment to make it oleophilic. The invention also concerns an additive in accordance with any of the foregoing claims for the biodegradation of hydrocarbons.

The present invention relates to a novel additive for use inbigdegradation processes, and to its application in the treatment ofmedia polluted by hydrocarbons, by accelerating natural bigdegradation.

Numerous processes exist using microorganism cultures grown undercontrolled conditions (in reaction vessels for example) for subsequentuse on the medium to be treated. Such processes are however ineffectivewhen there is a requirement to operate in an outside environment.Problems are observed relating to the microorganisms becoming diluted inthe natural medium, together with problems of competition withindigenous microorganisms which are much better adapted to the relevantmedium. There is thus a trend towards the solution consisting instimulating indigenous microorganisms by supplying them with elements ofnutrition necessary to their development and which constitute a limitingfactor in the natural medium.

Additionally, nutritional additives have been proposed for addition tothe medium to be treated. Such additives can be fertilizers of the typeemployed in agriculture, or synthetic proteinaceous products, or yetagain bacterial lyophilisates with the nutrient. These products satisfythe carbon, nitrogen and phosphorous requirements of the bacteria. Theparticular requirements of microorganisms for nitrogen and phosphorouscorrespond to an N/P molar ratio which can vary over a large rangewithout substantial alteration in effectiveness. Apart from nitrogen andphosphorous, these additives include assimilable carbon. Suchassimilable carbon is contained in hydrocarbon molecules the chain ofwhich is similar to an aliphatic chain encountered in the hydrocarbons.Moreover, these products act as a starter, in other words they favor thevery beginning of the reaction.

Availability of nutrients is also a significant problem as thisdetermines hydrocarbon degradation kinetics. In order to speed thingsup, various solutions have been proposed, consisting in mixing thenutrients with various additives and forming suspensions, andparticularly, emulsions. French patent 2,490,672 disclosesmicroemulsions in which the nutrient substances are in aqueous solutionwhich is put in microemulsion form in a lipid-miscible fluid. However,this technique implies a microemulsion-forming step and requires thepresence of additives such as surfactants and others, which areexpensive. French patent 2,512,057 discloses an improvement to thesolution proposed to the above-cited patent, which consists in providingthe source of nitrogen in a dual-system form comprising two differentchemical sorts of nitrogen compounds. A preferred system is a systemconsisting of urea and aminated acids. Moreover, this patent teachesthat aminated acids alone are not as effective as the dual system. Thisdual system is nevertheless a microemulsion and suffers from the samedisadvantages as all (micro)emulsions.

However, there are problems over toxicity with such synthetic additives,due to the presence of derivatives such as for example, butoxyethanol,and other similar products.

Thus, one looks for natural products to use as additives; but now theproblem is that these products do not contain carbon in a form which isclose to an aliphatic group of the type which is present in thehydrocarbons and, because of this, are not able to set up the desiredstarter effect. Moreover, one looks for additives which can be employedwithout the need for producing a (micro)emulsion or for expensiveadditives.

The use of meal of animal origin is further known, this being used as anutrient for microorganisms, the latter being in aqueous solution orsuspension.

Biosis Previews Databank, Philadelphia, Biosis Number 83042449, G.A.Kochkina et al.: "Development of a Nutrient Medium for CultivatingNtomophthora-Thaxteriana"& Biotekhnologiya, vol. 4, 1986, pages 46-51,discloses the use of fish meal as a nutrient for microorganism culture,for example Entomophthora-Thaxteriana fungi.

Biosis Previews Databank, Philadelphia, Biosis Number 70050436, M. Rusanet al.: "Influence of Animal Proteins on the Fermentation ofAntibiotics"& Bol Soc Broteriana 52, vol. 0, 1978, (Recd. 1979), pages29-36, describes the use of meat proteins or blood as a source ofnitrogen for producing antibiotics from fungus-type microorganisms.

Biosis Previews Databank, Philadelphia, Biosis Number 70043472, O. Yagiet al.: "Degradation of poly-chlorinated biphenyls by microorganisms"&J. Water Pollut Control Fed 52, vol. 5, 1980, pages 1035-1043, describesthe use of microorganisms for fighting pollution, meat extract beingadded to the culture medium.

However there is no indication that these same protein meals can also beemployed in a hydrocarbon-polluted medium, in other words one that isfar removed from a simple aqueous medium.

The applicant has found that, surprisingly, the additive according tothe present invention meets all the requirements stated above.

Thus, the present invention provides a biodegradationenhancing additivecharacterized in that it consists of a mixture comprising:

(i) at least one source of assimilable nitrogen consisting of at leastone unsubstituted or substituted aminated acid;

(ii) at least one source of phosphorous;

in a nitrogen/phosphorous (N/P) ratio of from 2 to 100; said additivehaving been subjected to a treatment designed to render it oleophilic.

The expression biodegradation should be taken to mean degradation by amicroorganism, which is either present in situ or brought from outside.This application can hence be carried out in an outdoors medium in thepresence of indigenous bacterial flora, or on the ground in the presenceof a specific added bacterial flora, if the existing flora is consideredinsufficient.

The microorganism employed can be a yeast, a fungus or a bacteria; infact, any microorganism able to break down a hydrocarbon is appropriate.The following can be cited by way of non-limiting examples: Pseudomonas,Acitenobacter, Flavobacterium, Artrobacter, Corynebacterium.

Assimilable nitrogen should be taken to mean nitrogen that iseffectively metabolized by the microorganism during degradation.

The stated treatment, which has the aim of rendering the additiveoleophilic (i.e. having an affinity for oils), can be a conventionaltreatment. The following can be cited as examples: acylation,esterification, grafting of a long radical onto various groups, a Schiffbase transformation, carbamate formation in the presence of isocyanate,and others. Preferably, the treatment consists of acylation. The carbonchain of the acyl group is preferably a fatty acid chain;advantageously, an acid chloride is employed, particularly of laurylicacid.

Aminated acids able to be employed in the framework of the presentinvention can consist of any aminated acid, whether natural orconsisting of closely related synthetic acids such as ornithine, andothers. These aminated acids can be substituted or unsubstituted. Whenthey are substituted, the substituent can be an alkyl, lower alcoxy orhydroxy group, and others as well. Preferably, the aminated acid isselected from the group comprising lysin, methionine, cystine,threonine, tryptophan, hydroxylysin, hydroxyproline, and mixturesthereof.

Preferably, the source of assimilable nitrogen makes up at least 5% byweight of the total weight of said biodegradation additive.

In one embodiment, the source of assimilable nitrogen is found inproteins which represent at least 50% by weight of the total weight ofthe said additive.

Any source of phosphorous, whether natural or synthetic, is appropriate.The preferred source of phosphorous is a mineral salt of phosphorous.

The said N/P ratio is advantageously comprised between 4 and 40 and ispreferably equal to about 16.

In one embodiment of the invention, the additive is an animal meal.

The meal can be a fish meal, or alternatively it can be a meal obtainedfrom meat.

Fish and meat meals are obtained by any conventional manufacturingmethod. By way of example, the following process can be cited for theproduction of meat meal: cutting up of animal carcasses followed bymilling, size grading, pre-heating, draining, drying, pressing and finalmilling. The following process can be cited as typical of the productionof fish meal: cutting up and cooking the fish product and pressing it,mixing it with a concentrate of liquid from pressing, and then drying,size-grading and finally milling it.

The composition of such meals can vary over a wide range; by way ofexample the following can be provided as examples which arerepresentative but non-limiting of the compositions for various meals:

Fish meal:

proteins: 60 to 85%

including the main aminated acids: lysin, methionine, threonine.

fatty matter: 3 to 25%.

inorganic matter (phosphorous, calcium, chlorides): 5 to 24%,

Meat meal:

proteins: 60 to 85%

including the main aminated acids: lysin, threonine, hydroxyproline.

fatty matter: 2 to 7%.

inorganic matter (phosphorous, calcium, chlorides): 7 to 28%.

The use of additives according to the present invention, such as meat orfish meals, is hence useful for biodegradation of hydrocarbons on theground, in sediments and on the surface of water. Sediments polluted byhydrocarbons can originate from accidental or non-accidental hydrocarbonspill-age, such as the cleaning of tanks, highways, land, etc. This useis just as suitable and profitable in the case of treatment in enclosedmedia such as reaction vessels, waste pits, hydrocarbon storage vessels,and so on.

The hydrocarbon-to-additive ratio is variable. The weight ratio ofadditive/hydrocarbons is generally comprised between 3 and 30.Preferably, the weight ratio is about 10.

The present invention also covers the use of the present additives forbiodegradation of hydrocarbons.

The following examples illustrate the invention in more detail, withouthowever limiting it.

EXAMPLES

Composition of additives

The composition of the biodegradation additives is given in the tablebelow.

    ______________________________________                                                    %        %                                                                    Nitrogen Phosphorous     % Carbon                                 Products    (N)      (P)        N/P  (C)                                      ______________________________________                                        fish meal   12.1     0.6        20.2 50.1                                     (Solatlante G)                                                                meat meal: 60%                                                                             9.8      3.95      2.5  35.4                                     (Viandor extraction)                                                          meat meal: 70%                                                                            12.0     3.1        3.9  40.8                                     (Viandor extraction                                                           greaves)                                                                      meat meal: 80%                                                                            12.9     1.4        9.2  40.5                                     (Viandor extraction                                                           greaves)                                                                      ______________________________________                                    

    ______________________________________                                                             MEAT    MEAT    MEAT                                                   FISH   60%     70%     80%                                      ______________________________________                                        PROTEINS        70-73    60      70    80                                     Lysin           5.20     3.25    4.20  4.86                                   Methionine      2.05     0.85    1.00  1.20                                   Cystine         0.55     0.85    0.42  0.48                                   Threonine       2.65     2.00    2.40  2.75                                   Tryptophan      0.70     0.45    0.70  1.10                                   Hydroxylysin    --       0.45    0.42  0.48                                   Hydroxyproline  --       3.42    3.85  4.40                                   MOISTURE        3-5      4-9     3-7   3-7                                    FATTY MATTER    20-23    2-6     2-4   2-4                                    INORGANIC MATTER                                                                              5-7      25-28   17-20  7-10                                  Phosphorous     0.40-0.80                                                                              3.5-4.2 2.9-3.3                                                                             1.2-1.6                                Calcium         0.15-0.50                                                                              7-9     5.5-7.0                                                                             2.4-3.7                                Chlorides (NaCl)                                                                              2-3      1.4-1.6 1.1-1.5                                                                             0.8-1.0                                ______________________________________                                    

Their average analytical composition was as follows:

The present invention is illustrated in greater detail in the examplesthat follow which should be considered as illustrative but not limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the evolution of ammoniacal nitrogen in reservoirsand reactors when the meal has and has not been acylated.

FIGS. 3 and 4 show the evolution of orthophosphate content in reservoirsand reactors when the meal has and has not been acylated.

FIGS. 5 and 6 show the salting-out of nitrogen in reactors when the mealhas and has not been acylated.

FIGS. 7 and 8 show the salting-out of phosphorous in reactors when themeal has and has not been acylated.

FIGS. 9 and 10 show the development over time of total andhydrocarbon-specific bacterial flora in the presence of non-acylatedmeal and acylated meal.

FIG. 11 shows the evolution of biodegradation index of alkanes for twotests: one in the presence of acylated meal and the other in thepresence of non-acylated meal.

FIGS. 12 and 13 show the development over time of total andhydrocarbon-specific bacterial flora in three ponds, one acting as acontrol, the others in the presence of acylated and non-acylated meal.

FIG. 14 shows the composition of samples of crude recovered from thethree ponds at time 0, and after 42 days, showing the alkane, aromatic,asphaltene and resin fractions.

FIGS. 15 to 18 are chromatograms of the alkane fraction for,respectively, Arabian light crude BAL 150 at day 0, the control pondafter 42 days, the pond treated with non-acylated meal after 42 days andthe pond treated with acylated meal after 42 days.

EXAMPLE 1

Biodegradation of hydrocarbons in the presence of acylated animal meals.

In order to render animal meals more oleophilic, crude meals weresubjected to an acylation reaction.

Synthesis of acylated meal.

Synthesis of the acylated meal was obtained under the conditionsdescribed below.

Synthesis was carried out in a solvent medium.

This reaction was based on bringing animal meal (fish meal) into contactwith an acid chloride (lauryl chloride C₁₂ H₂₃ C₁₀) in the presence of asolvent (dichloromethane CH₂ C₂). A proton acceptor consisting oftriethylamine [(C₂ H₅)₃ N] was added to the medium.

In a reaction vessel, the mixture consisting of lauryl chloride, fishmeal, dichloromethane and triethylamine was agitated (mechanicalagitation) for 24 hours at 30° C.

An excess amount of acid chloride (+20%) over that amount necessary toreact on the amine groups of the lysin was employed. Lysin representssome 5% of the fish meal proteins. The amount of triethylamine was addedin the same proportions.

After reacting for 24 hours, the acylated meal was washed over a filterwith a solvent (dichloromethane) to eliminate excess acid chloride. Thecake was then redissolved in water and filtered to eliminate excesstriethylamine and the salt formed. The cake consisting of the acylatedmeal was then kiln dried.

First test (Extent of oleophilic nature of acylated meal).

The extent of the oleophilic nature of crude meal or acylated meal wasmeasured using a distribution coefficient test.

A mixture of nitrogen-free artificial seawater (700 ml) + hydrocarbons(Arabian light crude BAL 150: 28g) and a known amount of animal meal onthe surface of the hydrocarbons was agitated for 5 minutes in aseparating funnel. After agitation, decantation was allowed to proceedfor 12 hours and the nitrogen content of the aqueous phase was measured.This nitrogen content is a reflection of how much of the nitrogencontained in the meal has passed into the aqueous phase. It is thuspossible to calculate how oleophilic the meal is. Table I below givesthe results obtained.

                                      TABLE I                                     __________________________________________________________________________    Extent of oleophilic nature of animal meals:                                  (HC: hydrocarbons. N: nitrogen)                                               __________________________________________________________________________                       Amount                                                                              Amount of                                            Animal  meal/HC                                                                            % of N in                                                                           of nitrogen                                                                         nitrogen in                                                                         % of nitrogen                                  meal    weight %                                                                           meal  introduced                                                                          the water                                                                           in the water                                   __________________________________________________________________________    Raw meal                                                                              3.54%                                                                              12.1% 120 mg                                                                              123.9 mg                                                                            100.0%                                         Acylated meal                                                                         3.54%                                                                              12.1% 120 mg                                                                               3.5 mg                                                                              2.9%                                          __________________________________________________________________________

It can thus be observed that subjecting the animal meal to acylationtreatment renders the meal oleophilic and that salting-out of nitrogendrops off distinctly when the meal has been acylated.

Second test (hydrocarbon biodegradation in the presence of acylated ornon-acylated meal).

In order to test the effectiveness of animal meals in hydrocarbonbiodegradation, a scintillometric measurement technique was used with aradioactive hydrocarbon (hexadecane) model. It is possible to followbiodegradation by monitoring 14_(CO2) production in line with thefollowing principle: In order to follow the breakdown kinetics of themarked substrate the amount of ¹⁴ CO₂ released by a bacterial culturewas observed. A technique employing a mini-reactor (5 ml) was used forthis, the reactor containing a bacterial culture (nutrient medium: 1 mland inoculum: 0.1 ml), the reactor being enclosed in a scintillationflask containing 2.5 ml molar soda. After incubation at 20° C. indarkness and without agitation the amount of ¹⁴ CO₂ trapped in the sodawas analyzed after acidifying the culture medium and after adding ascintillation liquid (Hionic fluoride). The same applied to the markedsubstrate remaining in the flask. Radioactivity was read on a BeckmannInstruments LS 3801 scintillation counter.

Table II below gives the results obtained.

                  TABLE II                                                        ______________________________________                                        Biodegradation (%) of hexadecane with and without the                         presence of acylated and non-acylated meal                                    Time (days)                                                                             Hexadecane  Raw meal  Acylated meal                                 ______________________________________                                         0        0.3%        0.0%       0.1%                                          4        0.0%        2.2%      45.9%                                         10        0.0%        9.1%      13.1%                                         20        0.0%        4.4%      33.0%                                         ______________________________________                                    

The biodegradation rate of hexadecane alone remained at 0. The bacteriapresent are not then capable of degrading hexadecane as such. However,in the presence of animal meal acceleration of hexadecane biodegradationwas observed. Acceleration was more pronounced when the meal wasacylated.

EXAMPLE 2

Biodegradation of hydrocarbons in the presence of acylated ornon-acylated animal meal, in an outdoor medium

In order to demonstrate the value of rendering animal meals oleophilic,tests were carried out in an outdoor medium to verify if the oleophilicnature of the meal made it possible to maintain the nutrient elements(nitrogen and phosphorous) in contact with the hydrocarbons thusspeeding up biodegradation.

Acylation of fish meal

The animal meals employed for examples 2 and 3 were fish meals. Thefollowing modifications were made to chemical synthesis compared withwhat is described above for example 1:

reaction temperature: this was of the order of 50° C, corresponding tothe solvent reflux temperature,

the duration of the reaction was 17 hours,

the acid chloride was in excess by 300% in correspondence to theparameter which underwent the biggest modification compared to thesynthesis in example 1.

The other parameters and test procedures were unchanged.

Test:

The test setup used in this series of experiments consisted of a reactorcontaining 100 ml of hydrocarbon-polluted (with 2.5 ml light Arabian)sea water. The acylated or non-acylated animal meal was applied to thesurface of the hydrocarbon in an amount of 10% by weight on the basis ofthe amount of hydrocarbon present. The reactor was continuously agitatedand aerated. The water in the reactor was renewed continuously duringthe 15 days with sea water contained in a reservoir, 8 renewals beingperformed per day. The effluent was collected at the outlet from thereactor and physical-chemical analyses (NH₄ ; NO₃ PO₄ ³⁻ hydrocarbons)and bacteriological analyses (total and specific bacteria) were carriedout. These same analyses were performed on the water in the reservoir.At the end of the test, the complete reactor was sacrificed and theremaining hydrocarbons were extracted using chloroform.

The physical-chemical analyses were carried out using the applicablestandards: AFNOR NF T90-015 for ammoniacal nitrogen, standard methods4500-NO3-E for nitrogen in nitrate form, standard method 4500-PC-Vn AcidCol. Meth. for orthophosphate.

Total bacteria were counted using the most probable number method, in aliquid medium (Marine Broth 2216). Hydrocarbon-specific bacteria werealso counted using the most probable number technique in a liquidmedium, in which the hydrocarbons represent the only source of carbon.The hydrocarbons were analysed by gaseous phase chromatography.

Two tests were done: one with non-acylated animal meal and the otherwith the same animal meal, but acylated this time. Each test lasted 15days. The results obtained are given below. The evolution of the mineralelements (NH₄₊ ; PO₄ ³⁻) are given in tables III and IV and in FIGS. 1,2, 3 and 4.

                  TABLE III                                                       ______________________________________                                        Evolution of ammoniacal nitrogen content in                                   the two tests: acylated and non-acylated meal                                 NON-ACYLATED MEAL     ACYLATED MEAL                                           N--NH4+ Reservoir  Reactor    Reservoir                                                                             Reactor                                 Days    mg/l       mg/l       mg/l    mg/l                                    ______________________________________                                        0       0.50       0.50       4.00    4.00                                    1       0.52       1.20                                                       2                             3.50    5.00                                    4       0.75       2.90       2.85    3.50                                    6       0.90       3.40                                                       7        1.025     3.50       2.00    1.10                                    8       1.15       0.50       1.50                                            11      0.50       0.40                                                       12      0.50       0.50                                                       13      0.50       0.49                                                       14       0.625     0.25               0.80                                    15      0.75       0.75                                                       16                            1.48    0.45                                    ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Evolution of orthophosphate content in the two tests:                         with acylated and non-acylated meal                                           NON-ACYLATED MEAL     ACYLATED MEAL                                           P--PO.sub.4.sup.3-                                                                    Reservoir  Reactor    Reservoir                                                                             Reactor                                 Days    mg/l       mg/l       mg/l    mg/l                                    ______________________________________                                        0       0.00       0.00       0.05    0.05                                    1        0.025     0.59                                                       2                              0.025  0.30                                    4       0.10       0.08               0.02                                    5                  0.00                                                       6        0.125     0.03                                                       7                  0.00       0.00    0.03                                    8       0.10        0.025                                                     9                             0.00    0.00                                    11      0.13       0.04                                                       12                 0.09                                                       13      0.20       0.10                                                       14                  0.085     0.00    0.00                                    15       0.110      0.110     0.00                                            16                                    0.04                                    ______________________________________                                    

The results obtained differed for acylated and non-acylated meal. It canbe observed that the acylated meal produced less salting-out of thenitrogen and phosphorous. Account should be taken of the comparisonbetween nitrogen and phosphorous concentrations in the water in thereservoir which were varying as this was "living" (plancton, bacteria,etc. . .) seawater. Availability of these results thus made it possibleto estimate what amount of nutrient had been eliminated by subtractionwith the concentrations measured in the reservoirs. The results aregiven in table V and in FIGS. 5, 6, 7 and 8.

                  TABLE V                                                         ______________________________________                                        Salting-out of nutrient: nitrogen and phosphorous                             in each reactor: with acylated and non-acylated meal                          AMMONIACAL NITROGEN  PHOSPHATE                                                     Non-acylated Acylated   Non-acylated                                                                           Acylated                                Days meal         meal       meal     meal                                    ______________________________________                                        0    0.000        0.000       0.000   0.000                                   1    0.680                    0.565                                           2                                     0.275                                   4    2.150        1.500      -0.020                                           5                 0.650                                                       6    2.500                   -0.095                                           7    2.475        -0.900              0.030                                   8    -0.650                  -0.075                                           9                                     0.000                                   10                                                                            11   -0.100                  -0.090                                           12   0.000                                                                    13   -0.010                  -0.100                                           14   -0.375                           0.000                                   15   0.000                    0.000                                           16                -1.030                                                      ______________________________________                                    

These results clearly show that salting-out was much less pronounced forthe meal rendered oleophilic by acylation. This treatment thus makes itpossible to attain a product that spends more time in contact with thehydrocarbon. Thus, those nutrients-- nitrogen and phosphorous-- that areindispensable to hydrocarbon-specific bacterial development are presentat the place where biodegradation is occurring, in other words at thewater-hydrocarbon interface. The results concerning bacterial floraevolution are given in table VI and VII, and in FIGS. 9 and 10.

                  TABLE VI                                                        ______________________________________                                        Total and hydrocarbon-specific bacterial flora evolution                      in reactor with non-acylated meal                                             RESERVOIR         REACTOR                                                             Total         Total    Specific                                               bacteria      bacteria bacteria                                       Days    Bact/ml       Bact/ml  Bact/ml                                        ______________________________________                                        0       5 · 10.sup.3                                                                       5 · 10.sup.3                                                                    5 · 10.sup.3                        4       8 · 10.sup.4                                                                         8 · 10.sup.5                                                                2.4 · 10.sup.5                        6       5 · 10.sup.5                                                                       1.1 · 10.sup.6                                                                5 · 10.sup.5                          7                                                                             8       2.2 · 10.sup.5                                                                     1.1 · 10.sup.6                                                                5 · 10.sup.5                          9                                                                             11      1 · 10.sup.5                                                                       1.1 · 10.sup.6                                                                5 · 10.sup.5                          13      8 · 10.sup.4                                                                       2.4 · 10.sup.5                                                                2.4 · 10.sup.5                        14                                                                            15      5 · 10.sup.4                                                                       1.3 · 10.sup.5                                                                2.6 · 10.sup.5                        16                                                                            ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        Total and hydrocarbon-specific bacterial flora evolution                      in reactor with acylated meal                                                 RESERVOIR         REACTOR                                                             Total         Total    Specific                                               bacteria      bacteria bacteria                                       Days    Bact/ml       Bact/ml  Bact/ml                                        ______________________________________                                        0       8 · 10.sup.3                                                                       8 · 10.sup.3                                                                  8 · 10.sup.3                          2       5 · 10.sup.4                                                                       2.2 · 10.sup.6                                                                8 · 10.sup.5                          4       2.2 · 10.sup.4                                                                     5 · 10.sup.5                                                                  2.4 · 10.sup.5                        7       1.2 · 10.sup.5                                                                     2.6 · 10.sup.6                                                                5 · 10.sup.6                          8                                                                             9       8 · 10.sup.4                                                                       5 · 10.sup.6                                                                  1 · 10.sup.6                          11                                                                            13                                                                            14      1.2 · 10.sup.5                                                                     5 · 10.sup.5                                                                  3 · 10.sup.6                          15                                                                            16      9 · 10.sup.4                                                                       4 · 10.sup.5                                                                  2.2 · 10.sup.5                        ______________________________________                                    

Bacterial counts carried out during the two tests made it possible tobring to light the fact that, in the case of the acylated meal,bacterial development was not only faster, but, above all, showed howmuch difference there was in the number of bacteria found in thereservoir (which entered the reactor every day) this being higher in thepresence of the meal. The latter thus has a favorable stimulating effecton total and hydrocarbon-specific bacteria, as the nutrients are moreavailable.

Hydrocarbon biodegradation quantification was carried out by estimatinga biodegradation index calculated starting from gaseous-phasechromatography. These indexes are the C17/pristane and C18/phytaneratios. The decrease of these ratios is correlated to the biodegradationof the aliphatic hydrocarbons. The ratios are given in table VIII.

                  TABLE VIII                                                      ______________________________________                                        Degree of biodegradation obtained                                             after 15 days                                                                 ______________________________________                                        NON-ACYLATED MEAL    ACYLATED MEAL                                                   C17/       C18/       C17/    C18/                                     Days   pristane   phytane    pristane                                                                              phytane                                  ______________________________________                                         0     4.4        2.5        4.4     2.5                                      15     4.4        2.5        3.8     2.3                                      ______________________________________                                    

The results, in particular the evolution of biodegradation indexesdemonstrate that the acylated meal gave better results than thenon-acylated meal.

The results make it possible to state that the acylation of the animalmeal leads to a product that stays in contact with the hydrocarbons:nitrogen and phosphorous are present at the water-hydrocarbon interfaceand this stimulates the bacterial flora, whether this be specific ortotal, and hydrocarbon biodegradation resulting therefrom is alsostimulated.

Acylated animal meal thus has an advantage over non-acylated meal inspeeding up hydrocarbon biodegradation.

EXAMPLE 3

Biodegradation of hydrocarbons in the presence of acylated andnon-acylated animal meal carried out outdoors on a large scale

In view of the results of the laboratory experiments which showed thevalue of employing an acylated animal meal, a trial on a much largerscale was carried out. The trial was done on 3 tanks of 400 literscapacity continuously supplied with fresh seawater pumped from a lagoonsituated next to the tanks. The renewal rate of the water in the tankswas 4 times their volume per day. Oil (Arabian light topped at 150° C.)was introduced into each tank (1 liter). One tank was kept as a controland no oil was introduced; non-acylated meal (5%/crude) was added to thesecond tank; acylated meal (5%/crude) was added to the third tank. Thenon-acylated and acylated meal was identical to that used in example 2.

Throughout the experiments, which lasted 2 months, total andhydrocarbon-specific bacterial flora was studied using the same testprocedure as the one described for example 2. The hydrocarbons were alsofollowed permanently.

The results of the bacteriological studies are given in table IX and inFIGS. 12 and 13.

                                      TABLE IX                                    __________________________________________________________________________    Total and specific bacteria count                                             in hydrocarbons                                                               Total bacteria         Specific bacteria                                      Bact./ml               Bact./ml                                                         Non-acylated                                                                         Acylated    Non-acylated                                                                         Acylated                                  Days                                                                              Control                                                                             meal   meal  Control                                                                             meal   meal                                      __________________________________________________________________________    0   2.4E + 06                                                                           2.4E + 06                                                                            2.4E + 06                                                                           4.6E + 03                                                                           1.1E + 06                                                                            4.6E + 05                                 1   2.4E + 06                                                                           2.4E + 07                                                                            2.4E + 07                                                                           1.1E + 04                                                                           1.1E + 06                                                                            4.6E + 05                                 3   2.4E + 06                                                                           2.4E + 06                                                                            2.4E + 06                                                                           1.1E + 04                                                                           4.6E + 05                                                                            1.4E + 07                                 5   1.5E + 06                                                                           9.3E + 06                                                                            9.3E + 07                                                                           1.4E + 05                                                                           1.4E + 07                                                                            1.4E + 07                                 8   2.3E + 06                                                                           4.0E + 06                                                                            2.3E + 06                                                                           1.8E + 05                                                                           1.6E + 07                                                                            2.0E + 07                                 14  3.1E + 06                                                                           6.9E + 06                                                                            2.3E + 07                                                                           1.8E + 06                                                                           2.0E + 07                                                                            2.8E + 07                                 22  2.5E + 07                                                                           3.9E + 07                                                                            4.3E + 06                                                                           1.4E + 07                                                                           4.6E + 07                                                                            7.9E + 07                                 42  2.5E + 08                                                                           4.6E + 08                                                                            6.3E + 07                                                                           2.4E + 07                                                                           2.4E + 08                                                                            5.0E + 08                                 __________________________________________________________________________

In the tanks treated with acylated or non-acylated meal, bacterial floradevelopment was more pronounced than in the control. The animal mealsthus had a stimulating effect on indigenous bacterial flora.

It was observed that development was greater in the tank treated withacylated meal than in the tank treated with non-acylated meal. Acylationof the meal makes it possible to keep the product close to thehydrocarbon layer thus favoring bacterial flora development.

The results obtained from hydrocarbon analyses are given in FIG. 14 andin the chromatograms of FIGS. 15 to 18.

An interpretation of the evolution of the various fractions of crudemade it possible to estimate if biodegradation had occurred. Thus, whereoil biodegradation had occurred, a reduction in the alkane and aromaticfraction was observed accompanied by an increase in asphaltene and resinfractions.

On FIG. 14, it can be seen that there was a decrease in alkane fractionin the 3 tanks after 42 days, but the increase in the asphaltene+ resinfraction is greater in the tank treated with acylated meal. This result,which demonstrates that biodegradation was greater in the tank treatedwith acylated meal is corroborated by the chromatograms given at the endof this document. It can in fact be observed that there is a distinctdropoff in the alkane fraction between 0 and 42 days and this reductionis greater for the tank that was treated with the acylated meal.

The complete set of results obtained after 42 days shows that thepresence of the acylated meal favors hydrocarbon biodegradation. Theresults are even more significant after a longer period, 42 days being aperiod of time considered fairly short for observing hydrocarbonbiodegradation.

Moreover, visual observation made it possible to state that the acylatedmeal did not lead to the hydrocarbons flowing to the bottom of the tank.

This trial makes it possible to highlight the value of treating ahydrocarbon slick or layer with acylated animal meal. The value of thisis even more significant in view of the fact that there are no or onlyfew biodegradation additives available for treating floating oil slicks.

What is claimed is:
 1. A biodegradation enhancing additive consisting ofa mixture comprising:(i) at least one source of assimilable nitrogenconsisting of at least one unsubstituted or substituted aminated acidselected from the group consisting of lysine, methionine, cystine,threonine, tryptophan, hydroxylysine, hydroxyproline, and mixturesthereof; (ii) at least one source of phosphorous;in anitrogen/phosphorous (N/P) ratio of from 2 to 100; said additive havingbeen subjected to a treatment designed to render said additiveoleophilic.
 2. The biodegradation-enhancing additive according to claim1, wherein said treatment consists of an acylation reaction.
 3. Thebiodegradation-enhancing additive according to claim 2, wherein saidacylation is carried out using laurylic acid chloride.
 4. Thebiodegradation-enhancing additive according to claim 1, wherein saidsource of assimilable nitrogen represents at least 5% by weight of thetotal weight of said additive.
 5. The biodegradation-enhancing additiveaccording to claim 14, wherein said source of assimilable nitrogenoccurs in proteins representing at least 50% by weight of the totalweight of said additive.
 6. The biodegradation-enhancing additiveaccording to claim 1, wherein said source of phosphorous is a mineralsalt of phosphorous.
 7. The biodegradation-enhancing additive accordingto claim 1, wherein said nitrogen/phosphorous ratio is comprised in therange of from 4 to 40..
 8. The biodegradation-enhancing additiveaccording to claim 7, wherein said nitrogen/phosphorous ratio is equalto about
 16. 9. A biodegradation-enhancing additive for thebiodegradation of hydrocarbons, said additive consisting of a mixturecomprising:(i) at least one source of assimilable nitrogen consisting ofat least one unsubstituted or substituted aminated acid; (ii) at leastone source of phosphorous;in a nitrogen/phosphorous (N/P) ratio of from2 to 100; said additive having been subjected to a treatment designed torender said additive oleophilic, and wherein said additive is an animalmeal.
 10. The biodegradation-enhancing additive according to claim 9,wherein said animal meal consists of a fish meal.
 11. Thebiodegradation-enhancing additive according to claim 9, wherein saidanimal meal is of meat origin.
 12. The biodegradation-enhancing additiveaccording to claim 9, wherein said treatment consists of an acylationreaction.
 13. The biodegradation-enhancing additive according to claim9, wherein said acylation is carried out using laurylic acid chloride.14. The biodegradation-enhancing additive according to claim 9, whereinsaid source of assimilable nitrogen represents at least by weight of thetotal weight of said additive.
 15. The biodegradation-enhancing additiveaccording to claim 1, wherein said source of assimilable nitrogen occursin proteins representing at least 50% by weight of the total weight ofsaid additive.
 16. The biodegradation-enhancing additive according toclaim 9, wherein said source of phosphorous is a mineral salt ofphosphorous.
 17. The biodegradation-enhancing additive according toclaim 9, wherein said nitrogen/phosphorous ratio is comprised in therange of from 4 to
 40. 18. The biodegradation-enhancing additiveaccording to claim 9, wherein said nitrogen/phosphorous ratio is equalto about
 16. 19. A process for the biodegradation of hydrocarbonscomprising the step of applying a biodegradation-enhancing additiveaccording to claims 1 or 9 to the surface of a hydrocarbon-pollutedmedium.
 20. The process according to claim 19, wherein said additive ispresent in a weight ratio of additive to hydrocarbon comprised between 3and
 30. 21. The process according to claim 20, wherein said additive ispresent in a weight ratio of additive to hydrocarbon equal to about 10.